Ethanol provides a favorable alternative to the use of fossil fuels for energy generation, and increased use of ethanol for fuel could reduce dependence on fossil fuels as well as decrease the accumulation of carbon dioxide in the atmosphere. In the United States, biological production of ethanol, principally by fermentation of grain starches and sugars by yeast, is over four billion liters per year. However, cellulosic biomass potentially provides a far more abundant source of ethanol. Cellulosic biomass represents the greatest carbohydrate resource on earth, and is fixed photosynthetically at a rate of about 1011 tons per year globally.
Conversion of cellulosic biomass to ethanol requires that the polysaccharides of the biomass first be hydrolyzed to fermentable monosaccharides. Cellulose is a polymer of glucose units, and, while hydrolysis of cellulose is more difficult than hydrolysis of starches, hydrolysis of cellulose yields glucose that is readily fermented by yeasts such as Saccharomyces cerevisiae and Kluyveromyces marxianus. However, cellulosic biomass comprises, in addition to cellulose, more complex and heterogeneous polymers collectively known as hemicellulose. Unlike cellulose, hemicellulose contains saccharides besides glucose—principally the pentose xylose, as well as the pentose arabinose and the hexoses glucose, galactose, and mannose. The pentose content of some cellulosic biomass may reach as high as 35% of the total carbohydrate content (see Rosenberg, Enzyme Microbiol Technol 2:185-193 (1980)). Moreover, in many industrial processes, hemicellulose is hydrolyzed to monosaccharides more efficiently than cellulose. Thus, 35-50% of the fermentable sugars obtained by enzymatic or chemical hydrolysis of cellulosic materials may be derived from hemicellulose, and much of this sugar may be in the form of xylose or arabinose (Harris et al., USDA Forest Products Laboratory General Technical Report FPL-45 (1985)).
Ideally, biological production of ethanol from cellulosic biomass would employ a natural organism capable of efficiently fermenting all five of the most abundant monosaccharides liberated by hydrolysis of cellulose and hemicellulose—glucose, galactose, mannose, xylose, and arabinose—as well as the disaccharide cellobiose produced by enzymatic digestion of cellulose. Even more ideally, such an organism would be able to hydrolyze resilient polymers such as cellulose or hemicellulose without the addition of exogenous enzymes or chemicals.
No such organism is presently known. In particular, while many yeasts will assimilate pentose sugars and hexose sugars, conversion of pentose- and hexose-containing cellulose or hemicellulose to ethanol by yeasts is problematic (see Jeffries & Kurtzman, Enzyme Microb Technol 16:922-932 (1994); Schneider, Crit Rev Biotechnol 9:1-40 (1989)). Fermentation of arabinose to ethanol is almost unknown (see Dien et al., Appl Biochem Biotechnol 57-58:233-42 (1996); McMillan & Boynton, Appl Biochem Biotechnol 45-46:569-84 (1994)). A few yeasts capable of fermenting xylose have been isolated, but their thermotolerance, ability to ferment xylose anaerobically, and their metabolism of hexose sugars are unsatisfactory (see Jeffries & Kurtzman, supra).
Yeasts of the genus Kluyveromyces—particularly thermotolerant strains—have many properties making them well-suited for biological production of ethanol (see Banat et al., World J. Microbiol Biotechnol 14:809-21 (1998); Singh et al., World J. Microbiol Biotechnol 14:823-34 (1998)). Kluyveromyces strains assimilate pentose sugars. However, efficient fermentation of hexoses by Kluyveromyces strains has not been described. A single report of high xylose production by K. marxianus has appeared (Margaritis & Bajpai, Appl Environ Microbiol 44:1039-41 (1982)), but ethanol production was under aerobic conditions and no subsequent report has verified these findings. Other publications report little (Banat et al., supra) or no (Boyle et al., Biotechnol Lett 19:49-51 (1997)) ethanol production from xylose by K. marxianus, even under aerobic conditions.
U.S. Pat. No. 4,472,501 describes a yeast called Kluyveromyces cellobiovorus with the ability to ferment xylose and cellobiose to ethanol, but subsequent analysis of this strain has shown that it does not belong to the genus Kluyveromyces, but rather is an isolate of Candida intermedia (see Molnar et al., Antonie Van Leeuwenhoek 70:67-78 (1996); Ando et al., Biosci Biotechnol Biochem 60:1063-9 (1996); Martini & Martini, Antonie Van Leeuwenhoek 61:57-60 (1992)).
Thus, no Kluyveromyces strain capable of hydrolyzing cellulose has been described. There is a need in the art for thermotolerant organisms capable of both hydrolyzing cellulosic materials, and of efficiently fermenting the hexoses found in hydrolysates to ethanol. The present invention meets these and other needs.